Multi-channel fluorescence microscopic imaging control system for automatic E-FRET imaging according to fluorescence probe

Abstract

The invention relates to a multichannel fluorescent microscopic imaging control system for automatic E-FRET imaging according to a fluorescent probe, which is applied to a multichannel fluorescent microscopic imaging system and performs automatic imaging matching according to optical characteristics of the fluorescent probe and the imaging system by inputting related information of the imaging system and the fluorescent probe. The method comprises the steps that fluorescence probes capable of imaging are combined in pairs to form donor-receptor pairs, then preliminary FRET imaging screening is carried out, for the donor-receptor pairs obtained through preliminary screening, a sample is provided by a user for an imaging experiment, FRET effect judgment and E-FRET parameter correction are carried out according to the imaging experiment, and the donor-receptor pairs capable of generating the FRET effect are determined; automatically generating a corresponding E-FRET shooting process and storing the E-FRET shooting process; and when a user selects the stored donor-receptor pair and provides an experimental sample for an experiment, controlling an imaging system to execute an E-FRET shooting process corresponding to the donor-receptor pair to perform an imaging experiment on the experimental sample, and calculating an E-FRET result by using an E-FRET parameter correction result.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescence microscopic imaging, and in particular to a multi-channel fluorescence microscopic imaging control system based on automatic E-FRET imaging of fluorescent probes. Background Art

[0002] Multi-channel fluorescence microscopy is a highly specialized microscopy technique that allows researchers to simultaneously image multiple fluorescent markers in biological samples accurately, clearly, and without interference. The core of this technology is its ability to distinguish and record multiple independent fluorescence signals, thereby revealing complex cell structures, molecular interactions, and dynamic biological processes. A multi-channel fluorescence microscopy system usually includes a light source (including a light source and excitation filters), an optical device group (including emission filters, dichroic mirrors, etc.), a detector (CCD, photomultiplier tube, CMOS, etc.), a motorized stage, a control board, etc.

[0003] The current multi-channel fluorescence microscopy system supports the artificial selection of light sources and filter blocks / groups based on fluorescent probes to achieve imaging. For this type of microscopy system, researchers can only select fluorescent dyes that have been preset in the system, that is, fluorescent probes, for imaging. If the system is not preset, researchers need to compare the spectra of new dyes with existing dyes. Only if they have similar spectra with existing dyes can imaging be completed. If not, imaging cannot be completed. Moreover, the algorithm for matching imaging based on fluorescent probes in existing imaging systems is unknown. With the rapid development of the field of fluorescent probes in recent years, the types of fluorescent probes have increased rapidly, and the above-mentioned fluorescence microscopy system is very limited.

[0004] Fluorescence resonance energy transfer (FRET)

[0005] As a spectral nanomolecular ruler, it can achieve nanometer-level spatial resolution, and under the irradiation of specific light, the energy transfer characteristics between two fluorescent dyes can monitor the sample in real time. This feature makes it an important research tool in the fields of biology and chemistry.

[0006] The E-FRET method (i.e., channel-based sensitized emission method) is a non-destructive three-channel FRET apparent efficiency measurement method. Due to its rapid and non-destructive characteristics, the E-FRET method has become the most widely used method for dynamic monitoring of living cells, and it can better correct spectral crosstalk and system parameters. The E-FRET method involves two different fluorescent probes. For these two probes, first, each of them must be able to be imaged in the imaging system, and second, these two fluorescent probes must be able to produce a FRET effect. Before conducting an E-FRET experiment, it is necessary to use standard samples to pre-calibrate the optical detection system and the optical properties of the fluorescent group (i.e., parameter calibration), and once the calibration is completed, no system parameters can be changed in subsequent experiments. There is a high technical threshold, and the current microscope control system on the market cannot directly perform E-FRET imaging, which greatly limits the development and promotion of this technology.

[0007] The overall purpose of this project is to improve the multi-channel fluorescence microscopy imaging system so that it can automatically identify fluorescent probe pairs that can perform E-FRET imaging and quickly perform E-FRET automatic imaging. Summary of the invention

[0008] The purpose of the present invention is to provide a multi-channel fluorescence microscopic imaging control system based on automatic E-FRET imaging of fluorescent probes.

[0009] The object of the present invention is achieved through the following technical solution: a multi-channel fluorescence microscopy imaging control system based on automatic E-FRET imaging of fluorescent probes, which is applied to a multi-channel fluorescence microscopy imaging system, characterized in that it includes:

[0010] System parameter input software module: determining the relevant information required for establishing connection with each electric control in the imaging system and inputting the optical characteristic parameters of each optical element in the imaging system;

[0011] Fluorescent probe information input software module: input the optical characteristic parameters of the fluorescent probe;

[0012] Imaging matching software module: performing automatic imaging matching according to the optical characteristics of the fluorescent probe and the imaging system, and saving the imaging settings of the fluorescent probe;

[0013] Preliminary FRET imaging screening software module: for the fluorescent probes capable of imaging, two of them are combined into donor-acceptor pairs and then preliminary FRET imaging screening is performed;

[0014] FRET effect judgment and E-FRET parameter correction software module: for the donor-acceptor pairs preliminarily screened, a standard E-FRET imaging setting is generated (after clarifying the donor, the acceptor and their respective imaging settings (the respective imaging settings of the fluorescent probes are clarified by the imaging matching software module), the standard E-FRET imaging setting for the donor-acceptor pair can be clarified), and then the imaging system is controlled according to the standard E-FRET imaging setting to perform imaging experiments on the standard samples provided by the user, and the FRET effect is judged (judged according to the specific imaging situation) and the E-FRET parameter correction is performed accordingly, and for the donor-acceptor pairs determined to be capable of FRET effect, the corresponding E-FRET shooting process is automatically generated and saved; the standard samples include single-transfer donor samples, single-transfer acceptor samples and samples containing both donors and acceptors;

[0015] E-FRET imaging execution software module: when the user selects a saved donor-acceptor pair and provides an experimental sample for an experiment, the imaging system is controlled to execute the E-FRET shooting process corresponding to the donor-acceptor pair to perform an imaging experiment on the experimental sample, and the E-FRET result is calculated using the result of the E-FRET parameter correction.

[0016] The control system of the present invention can automatically identify fluorescent probes capable of imaging and combine them into donor-acceptor pairs capable of producing FRET effects, and generate corresponding E-FRET shooting processes. Users only need to select the corresponding donor-acceptor pairs and provide experimental samples to achieve automatic E-FRET imaging of the corresponding FRET pairs.

[0017] Perform a preliminary FRET imaging screen as follows:

[0018] 1) According to the normalized spectral characteristics of the fluorescent probe absorption spectrum , calculate the total area of ​​its absorption spectrum ; According to the normalized spectral characteristics of its emission spectrum , and the wavelength passband of the detection channel described in the imaging setup , calculate the total area of ​​its emission spectrum and the detectable wavelengths occupy the total area of ​​the emission spectrum ;

[0019] 2) If the donor-acceptor pair meets the following two conditions, the pair of fluorescent probes is considered to be a possible FRET pair:

[0020] ① For both the donor and the acceptor, the ratio of the total area of ​​the detectable band to the total area of ​​the complete emission spectrum must be greater than or equal to the threshold ,Right now

[0021]

[0022] Threshold It is set empirically to ensure that the emitted light of the fluorescent probe can be detected by the imaging system, that is, to define whether the fluorescent probe can be illuminated in the imaging system.

[0023] ② For the acceptor, the ratio of the area of ​​the overlapping part of its absorption spectrum and the donor emission spectrum to the total area of ​​the donor emission spectrum, as well as the ratio of the area of ​​the overlapping part of its absorption spectrum and the donor emission spectrum to the total area of ​​the acceptor absorption spectrum, must be greater than or equal to the threshold ,Right now , , Threshold Generally, it is taken as 0.3.

[0024] In the present invention, performing E-FRET parameter correction mainly refers to calculating E-FRET correction coefficients a, b, c, d, G, k, which includes the following steps:

[0025] Calculate the corrected spectral crosstalk coefficients a, b, c, and d of the single-turn donor and single-turn acceptor, where a represents the degree of crosstalk caused by the direct excitation of the acceptor by the donor excitation light, b represents the degree of crosstalk of the emission light of the acceptor directly excites the acceptor by the acceptor excitation light, c represents the degree of crosstalk caused by the direct excitation of the donor by the acceptor excitation light, and d represents the degree of crosstalk of the emission light of the donor directly excites the donor by the donor excitation light. Their calculation formulas are:

[0026]

[0027]

[0028] The theoretical sensitized emission value Fc is calculated using a sample containing both donor and acceptor:

[0029] , It represents the gray value of the detected image. Its subscripts have the following meanings: the first letter represents the excitation light, where D represents the donor excitation light and A represents the acceptor excitation light. The second letter represents the detection channel, where D represents the donor detection channel and A represents the acceptor detection channel. The letters in brackets represent the sample type, where D represents a single-transfer donor and A represents a single-transfer acceptor. DA represents that the sample contains both donors and acceptors, for example Indicates that when the sample is a single-transduction acceptor, the gray value of the image detected when the acceptor is excited by the donor excitation light;

[0030] Then the sensitization-quenching conversion factor G and the concentration correction factor k are calculated. The sensitization-quenching conversion factor G is defined as the ratio of the sensitized emission Fc in the DD channel after acceptor photobleaching to the corresponding donor recovery amount. The concentration correction factor k represents the ratio of the donor / acceptor fluorescence intensities of equimolar concentrations in the absence of FRET. The calculation formula is:

[0031]

[0032]

[0033] Where E is the theoretical FRET efficiency of a sample containing both donor and acceptor.

[0034] When automatic imaging matching is performed according to the optical characteristics of the fluorescent probe and the imaging system, an imaging channel is selected according to the brightness of the fluorescent probe in the detection channel of the imaging system, and the imaging channel is selected with a brightness greater than a set threshold The detection channel with the largest brightness is preferably used as the imaging channel.

[0035] In the present invention, the detection channel consists of an excitation light path and a detection light path. The excitation light path refers to the light path experienced during the excitation process from the light source to the sample, and the detection light path refers to the light path experienced by the emission light generated after the sample is excited to the detector.

[0036] The brightness of the fluorescent probe in the detection channel of the imaging system is determined by:

[0037] Calculate the wavelength passband of the excitation light path of the detection channel ;

[0038] Calculate the wavelength passband of the detection optical path of the detection channel ;

[0039] Calculate the extinction coefficient of the excitation light path corrected according to the excitation light source band of the detection channel and the photon utilization rate of the detection optical path , which are defined as:

[0040] ,

[0041] ,

[0042] in, is the molar extinction coefficient of the fluorescent probe; is the normalized spectral characteristic of the emission spectrum of the excitation light source; represents the normalized spectral characteristics of the fluorescent probe absorption spectrum; Indicates the transmittance of the objective lens of the detection channel to light; represents the quantum yield of the fluorescent probe; represents the normalized spectral characteristics of the emission spectrum of the fluorescent probe; The quantum efficiency of the detector of the detection channel to light;

[0043] Then calculate the brightness of the fluorescent probe in the detection channel , defined as:

[0044] .

[0045] Before calculating the brightness of the detection channel, it is recommended to select the excitation light source band of the detection channel in the following way:

[0046] Calculate the absorption spectrum peak of the fluorescent probe The peak of the emission spectrum of the excitation light source of the imaging system The difference and set the threshold Compare and choose The corresponding light source emits light band; the smallest one is preferred The corresponding light source emits light band.

[0047] Beneficial effects:

[0048] 1) The control system of the present invention can automatically identify the fluorescent probes that can be imaged to form a donor-acceptor pair that can produce a FRET effect, and generate a corresponding E-FRET shooting process. The user only needs to select the corresponding donor-acceptor pair and provide the experimental sample to achieve automatic E-FRET imaging of the corresponding FRET pair;

[0049] 2) The present invention also provides a solution for matching imaging channels according to fluorescent probes, and provides a preferred method for calculating channel brightness. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 is a control flow chart of the control system of the present invention;

[0051] Figure 2 It is a schematic diagram of the optical path of a dual-channel fluorescence microscopy imaging system;

[0052] Figure 3 This is a real-life image of a single-transfected EGFP sample (after contrast enhancement);

[0053] Figure 4 This is a real-life image of a single-transfected mCherry sample (after contrast enhancement);

[0054] Figure 5Three images were taken of a sample containing both GFP and mCherry according to the standard E-FRET protocol. DETAILED DESCRIPTION

[0055] The present invention aims to provide a multi-channel fluorescence microscopy imaging control system based on automatic E-FRET imaging of fluorescent probes. The preferred embodiment of the present invention is described in detail below in conjunction with the specific embodiments of the present invention.

[0056] Embodiment 1

[0057] A multi-channel fluorescence microscopic imaging control system for automatic E-FRET imaging based on fluorescent probes is applied to a multi-channel fluorescence microscopic imaging system, comprising a system parameter input software module, a fluorescent probe information input software module, an imaging matching software module, a preliminary FRET imaging screening software module, a FRET effect judgment and E-FRET parameter correction software module and an E-FRET imaging execution software module.

[0058] System parameter input software module: determines the relevant information of the imaging system (specifically including the communication protocol of each electric control in the imaging system so as to transmit information with the imaging system) and inputs the optical characteristic parameters of each optical element in the imaging system, specifically including:

[0059] (1.1) Input the emission spectrum of the multi-band light source of the imaging system. And the peak , i represents the i-th emission spectrum, and the normalized spectral characteristics of each emission spectrum are determined at the same time , and determine the excitation filter corresponding to each emission light Passband and stopband ;

[0060] (1.2) Record the normalized spectral characteristics of the optical elements in the optical device group in the imaging system: Determine the emission filter Passband and stopband , j represents the jth emission filter; determine the dichroic mirror Transmission band and reflective tape ; k represents the kth dichroic mirror.

[0061] (1.3) Enter the transmittance of the objective lens in the imaging system for light of different wavelengths ;

[0062] (1.4) Enter the number of the CMOS detectors for each channel in the imaging system , m represents the mth channel, which determines the quantum efficiency of each CMOS for light of different wavelengths .

[0063] The wavelength range considered by the imaging system of this embodiment is 300nm-800nm. All entered wavelength range values ​​are corrected. If the entered relevant data exceeds this range, it is discarded. If data is missing in a certain section of this range, it is uniformly considered to be a stop band or the light intensity is zero.

[0064] Fluorescent probe information input software module: input the optical characteristic parameters of the fluorescent probe, such as inputting the fluorescent probe Related information, n represents the nth fluorescent probe, including:

[0065] Fluorescent probes Peak value of absorption spectrum , the normalized spectral characteristics of the absorption spectrum ; The peak of the emission spectrum , the normalized spectral characteristics of the emission spectrum ; Molar extinction coefficient of fluorescent probe ; Quantum yield of fluorescent probe .

[0066] Imaging matching software module: performs automatic imaging matching according to the optical characteristics of the fluorescent probe and the imaging system, and saves the imaging settings of the fluorescent probe, specifically:

[0067] (3.1) Determine the wavelength band of the light source used for imaging of each fluorescent probe.

[0068] For a fluorescent probe , first calculate the fluorescent probes The absorption spectrum peak The peak value of all emission spectra of the light source The difference , i represents the difference with the peak value of the i-th emission spectrum of the light source. At the same time, a threshold is set ,when When the fluorescent probe Can be activated in this system, it is recommended to select the smallest one from the activatable options The corresponding light source emission spectrum peak The corresponding light source emits light in the band The excitation light When the fluorescent probe Cannot be triggered in this system.

[0069] (3.2) For fluorescent probes that can be excited, calculate the brightness of the fluorescent probe in each detection channel, and then select the imaging channel of the probe based on the brightness.

[0070] After the wavelength band of the light source is determined, for a certain fluorescent probe, there is an excitation light path for the fluorescent probe in the multi-channel fluorescence microscopy imaging system. and multiple detection light paths , m represents the mth detection optical path (the same as the number of detectors), the optical elements of the excitation optical path usually include a light source (excitation light and excitation filter), an objective lens (no light filtering), and some also include a dichroic mirror, and the optical elements of each detection optical path usually include an optical device group (emission filter and dichroic mirror), an objective lens (no light filtering), a reflector (no light filtering), etc. The detection channel consists of a certain laser optical path and a certain detection optical path.

[0071] In a multi-channel fluorescence microscopy system, for the excitation light path and each detection light path, considering the optical properties of multiple optical elements in the detection path, it is necessary to correct the extinction coefficient and quantum yield of the fluorescent probe.

[0072] First, the wavelength passband of each detection path is calculated.

[0073] For the excitation light path The optical components involved in the calculation include the light source (excitation light and excitation filter) and possibly a dichroic mirror (optical devices that do not filter light have no effect on the wavelength passband, so they are not considered in the following calculations). and excitation filter wavelength passband And the transmission band (or reflection band) of the dichroic mirror are intersected (if the dichroic mirror is included) to obtain the wavelength passband of the excitation light path , the calculation formula is:

[0074] (Without dichroic mirror)

[0075] (Dichroic mirrors are all transmissive)

[0076] (Dichroic mirrors are all reflective)

[0077] (Dichroic mirrors are both transmissive and reflective)

[0078] The i in the above formulas is determined by (3.1).

[0079] For the detection light path The optical elements involved in the calculation include multiple emission filters and multiple dichroic mirrors. When the detection light path is determined, that is, a is determined, all the optical elements in the light path and the filtering conditions of the elements are determined (that is, j and k have multiple values ​​and the values ​​are determined). The wavelength passband of all emission filters and the transmission band (or reflection band) of all dichroic mirrors are intersected to obtain the wavelength passband of the detection light path. , the calculation formula is:

[0080] (Dichroic mirrors are all transmissive)

[0081] (Dichroic mirrors are all reflective)

[0082] (Dichroic mirrors are both transmissive and reflective).

[0083] Determine the wavelength passband of the excitation light path and the wavelength passband of all detection optical paths Finally, due to the different absorption and emission characteristics of fluorescent probes for light of different wavelengths, the receiving characteristics of the SCMOS photosensitive area, and the different transmittances of various optical components in the system for light of different wavelengths, it is impossible to directly use the original extinction coefficient and quantum yield of the fluorescent probe for calculation. It is necessary to correct them and calculate the extinction coefficient of the excitation light path of the entire imaging system (including the cell sample transfected with the fluorescent probe) during imaging (the absorption capacity of the excitation light, defined as , i corresponds to the excitation light band, n corresponds to the fluorescent probe number) and the photon utilization rate of each detection light path (the ratio of the number of photons absorbed by the fluorescent protein to the number of photons that the SCMOS can finally receive, defined as , m corresponds to the detection channel number, n corresponds to the fluorescent probe number).

[0084] (i is unique and determined by (3.1))

[0085]

[0086] Fluorescent probes The brightness in each detection light path , defined as:

[0087] (i is unique and determined by (3.1))

[0088] Determine the imaging channels in which the fluorescent probe can be imaged, automatically match the corresponding detector number and save all the above settings for one-click imaging.

[0089] Set brightness threshold , and obtain the fluorescent probe The actual brightness in each detection light path After that, if , then the fluorescent probe cannot be imaged in this fluorescence microscopy imaging system; if there is only one m value such that , then the fluorescent probe Only one imaging channel can be imaged. The system automatically saves the number of the imaging channel and the corresponding detector number in the record of the fluorescent probe. After saving, one-click imaging can be performed. If there are multiple m values, , then the fluorescent probe Capable of imaging in multiple imaging channels, the system will take the maximum brightness value greater than the brightness threshold and use this imaging channel as a fluorescent probe The best imaging channel is selected, and the number of the imaging channel and the corresponding detector number are automatically saved in the record of the fluorescent probe and saved so that one-click imaging can be performed.

[0090] Preliminary FRET imaging screening software module: For the fluorescent probes capable of imaging, two of them are combined into donor-acceptor pairs and then preliminary FRET imaging screening is performed.

[0091] Perform a preliminary FRET imaging screen as follows:

[0092] 1) According to the normalized spectral characteristics of the fluorescent probe absorption spectrum , calculate the total area of ​​its absorption spectrum ; According to the normalized spectral characteristics of its emission spectrum , and the wavelength passband of the detection channel described in the imaging setup , calculate the total area of ​​its emission spectrum and the detectable wavelengths occupy the total area of ​​the emission spectrum ;

[0093] 2) If the donor-acceptor pair meets the following two conditions, the pair of fluorescent probes is considered to be a possible FRET pair:

[0094] ① For both the donor and the acceptor, the ratio of the total area of ​​the detectable band to the total area of ​​the complete emission spectrum must be greater than or equal to the threshold ,Right now

[0095]

[0096] Threshold It is set empirically to ensure that the emitted light of the fluorescent probe can be detected by the imaging system, that is, to define whether the fluorescent probe can be illuminated in the imaging system.

[0097] ② For the acceptor, the ratio of the area of ​​the overlapping part of its absorption spectrum and the donor emission spectrum to the total area of ​​the donor emission spectrum, as well as the ratio of the area of ​​the overlapping part of its absorption spectrum and the donor emission spectrum to the total area of ​​the acceptor absorption spectrum, must be greater than or equal to the threshold ,Right now

[0098]

[0099]

[0100] According to the literature, the threshold Typically set to 0.3.

[0101] FRET effect judgment and E-FRET parameter correction software module: for the donor-acceptor pairs preliminarily screened, a standard E-FRET imaging setting is generated, and then the imaging system is controlled according to the standard E-FRET imaging setting to perform imaging experiments on the standard samples provided by the user, and the FRET effect is judged (judged according to the specific imaging situation) and the E-FRET parameter correction is performed. For the donor-acceptor pairs determined to be able to produce the FRET effect, the corresponding E-FRET shooting process (including light source timing control, camera timing control, and imaging channel selection) is automatically generated; the standard samples include single-transfer donor samples, single-transfer acceptor samples, and samples containing both donors and acceptors.

[0102] For the possible FRET pairs screened out, the user needs to provide single-transfection samples of two fluorescent probes and samples containing both donors and acceptors for imaging experiments, obtain real images of several different experimental samples in the imaging system according to the generated standard E-FRET imaging settings, and then perform FRET effect judgment and E-FRET parameter correction based on these images. It should be noted that the samples containing both donors and acceptors need to have a certain theoretical FRET efficiency value E, which is provided by the user to the imaging system.

[0103] The standard E-FRET imaging setting is generated in the following manner: the FRET effect judgment and E-FRET parameter correction software module presets the E-FRET imaging template. The imaging matching software module above has determined the detection channels (including the detection channel number and the detector number) when the donor and the acceptor are imaged separately, as well as the optimal excitation band (for the light source in the imaging system) when they are imaged separately, and saves the above two types of information. Here, the FRET effect judgment and E-FRET parameter correction software module fills the information of the optimal excitation band of the donor fluorescent probe and the detection channels of the donor and the acceptor into the E-FRET imaging template, and fills the information of the optimal excitation band of the acceptor fluorescent probe and the detection channel of the acceptor into the E-FRET imaging template. After the saving is completed, when the user selects the donor-acceptor pair later, the system will automatically perform one-click E-FRET imaging on all samples of the donor-acceptor pair (including single-transfer donor samples, single-transfer acceptor samples, and samples containing both donors and acceptors) according to the saved E-FRET imaging settings, complete the E-FRET experiment, and perform FRET effect judgment and E-FRET parameter correction.

[0104] Standard E-FRET imaging specifically requires that the sample be excited with donor excitation light and imaged in two channels (donor and acceptor detection channels), and that the sample be excited with acceptor excitation light and imaged in the acceptor detection channel.

[0105] Regarding E-FRET parameter calibration, the E-FRET method first needs to use a single-turn donor and a single-turn acceptor to calibrate the spectral crosstalk coefficients a, b, c, and d; where a represents the degree of crosstalk caused by the donor excitation light directly exciting the acceptor, b represents the degree of crosstalk to the donor channel caused by the acceptor excitation light directly exciting the acceptor, c represents the degree of crosstalk caused by the acceptor excitation light directly exciting the donor, and d represents the degree of crosstalk to the acceptor channel caused by the donor excitation light directly exciting the donor. The calculation formula is:

[0106]

[0107]

[0108] When the sample is a single-transfection acceptor, the grayscale value of the image detected by the acceptor detection channel is excited by the donor excitation light. The first letter in the subscript DA (A) represents the excitation light (D represents the donor excitation light, A represents the acceptor excitation light), the second letter represents the detection channel (D represents the donor detection channel, A represents the acceptor detection channel), and the letters in the brackets represent the sample type (D represents a single-transfection donor, A represents a single-transfection acceptor, and DA represents that the sample contains both donors and acceptors). The rest are similar.

[0109] After obtaining the spectral crosstalk coefficient of E-FRET, the theoretical sensitized emission value Fc is calculated using a sample containing both the donor and the acceptor:

[0110] .

[0111] The theoretical FRET efficiency of a sample containing both donors and acceptors is E. After obtaining the values ​​of the spectral crosstalk coefficients a, b, c, d and the sensitized emission Fc, the sensitized quenching conversion factor G and the concentration correction factor k are calculated. The sensitized quenching conversion factor G is defined as the ratio of the sensitized emission Fc in the DD channel after acceptor photobleaching to the corresponding donor recovery amount, and the concentration correction factor k represents the ratio of the donor / acceptor fluorescence intensities of equimolar concentrations in the absence of FRET. The calculation formula is:

[0112]

[0113]

[0114] E-FRET imaging execution software module: when the user selects a saved donor-acceptor pair and provides an experimental sample for an experiment, the imaging system is controlled to execute the E-FRET shooting process corresponding to the donor-acceptor pair to perform an imaging experiment on the experimental sample, and the E-FRET result is calculated using the result of the E-FRET parameter correction.

[0115] After all E-FRET correction parameters are calculated, the E-FRET parameter configuration is saved according to the name of the current FRET pair, and the corresponding E-FRET shooting process is automatically generated. After the user selects the FRET pair according to the name, one-click E-FRET imaging and calculation of E-TRET results can be performed based on these 6 parameters and the previously generated E-FRET imaging settings. The control flow chart of the control system is shown in the figure. Figure 1 shown.

[0116] Below Figure 2 Taking the dual-channel fluorescence microscopy imaging system as an example, the implementation process of the control system of the above embodiment 1 is specifically introduced.

[0117] Figure 2 Introduction to the optical components of the dual-channel fluorescence microscopy system:

[0118] Light Source is a fully electrically controllable six-band LED light source that can emit six types of excitation light with peak wavelengths of 385nm, 435nm, 488nm, 515nm, 560nm, and 630nm;

[0119] EX is the excitation filter:

[0120] For the excitation light with a peak value of 385 nm, no excitation filter is provided;

[0121] For the excitation light with a peak value of 435nm, an excitation filter is set;

[0122] For the excitation light with a peak value of 488nm, an excitation filter is set;

[0123] For the excitation light with a peak value of 515nm, an excitation filter is set;

[0124] For the excitation light with a peak value of 560nm, an excitation filter is set;

[0125] For the excitation light with a peak value of 630nm, an excitation filter is set;

[0126] Objective is the objective lens, which is an Olympus 60x oil lens. The samples observed in this example are cells transfected with four fluorescent probes: EGFP, mcherry, EYFP, and ECFP.

[0127] D1 is a dichroic mirror, used for light splitting;

[0128] EM1 is the emission filter;

[0129] M1 is a reflector, which is used to reflect the light beam, and the loss is not considered;

[0130] D2 is a dichroic mirror, used for light splitting;

[0131] EM2 is the emission filter;

[0132] EM3 is the emission filter;

[0133] Both SCMOS1 and SCMOS2 are Hamamatsu cameras.

[0134] After connecting all the hardware facilities and setting up the system, follow the process in Implementation 1:

[0135] 1) Determine the relevant information of the imaging system and enter the optical characteristic parameters of each optical element in the imaging system (only considering the 300nm-800nm ​​band).

[0136] According to the optical information files of the light source, filter, and dichroic mirror, the normalized spectral characteristics of the six bands of the multi-band LED light source, the passband and stopband of the excitation filter corresponding to each band, and the normalized spectral characteristics of the optical elements in the optical device group are recorded. For the objective lens, the model is Olympus 60x oil objective, and the transmittance of this model of objective lens for light of different wavelengths is recorded.

[0137] For the two SCMOSs, their quantum yields for light of different wavelengths are recorded respectively.

[0138] Correct the wavelength bands involved in the optical characteristics of all the optical components recorded above, and fix the wavelength band range between 300-800nm. Take the excitation filter as an example. When recording, only the passband of 427nm-445nm is recorded. The system will automatically add the stopband of the excitation filter to 300nm-426nm, 446nm-800nm. The same applies to other optical components.

[0139] 2) Enter the optical characteristic parameters of the fluorescent probe.

[0140] There are four fluorescent probes used in this imaging system, namely mCherry, EGFP, EYFP, and ECFP.

[0141] For mCherry, the peak of its absorption spectrum was recorded as 587 nm, and the spectral characteristics of the absorption spectrum were recorded; the peak of its emission spectrum was recorded as 610 nm, and the spectral characteristics of the emission spectrum were recorded; the molar extinction coefficient was recorded as 72000; and the quantum yield was recorded as 0.22.

[0142] For EGFP, the peak value of its absorption spectrum was recorded as 488 nm, and the spectral characteristics of the absorption spectrum were recorded; the peak value of its emission spectrum was recorded as 507 nm, and the spectral characteristics of the emission spectrum were recorded; the molar extinction coefficient was recorded as 56000; and the quantum yield was recorded as 0.60.

[0143] For EYFP, the peak value of its absorption spectrum was recorded as 513 nm, and the spectral characteristics of the absorption spectrum were recorded; the peak value of its emission spectrum was recorded as 527 nm, and the spectral characteristics of the emission spectrum were recorded; the molar extinction coefficient was recorded as 83400; and the quantum yield was recorded as 0.61.

[0144] For ECFP, the peak value of its absorption spectrum was recorded as 434 nm, and the spectral characteristics of the absorption spectrum were recorded; the peak value of its emission spectrum was recorded as 477 nm, and the spectral characteristics of the emission spectrum were recorded; the molar extinction coefficient was recorded as 32500; and the quantum yield was recorded as 0.4.

[0145] 3) Automatically performing imaging matching according to the optical characteristics of the fluorescent probe and the imaging system, and saving the imaging settings of the fluorescent probe.

[0146] Determine the wavelength band of the light source used for imaging each fluorescent probe.

[0147] Set the difference threshold to 30.

[0148] For mCherry, the peak of its absorption spectrum is 587nm, which has a minimum difference of 27 with the peak of the excitation light in the 560nm band of the light source, and is less than the threshold of 30. The system determines that mCherry can be excited in the system and the peak of the excitation light band is 560nm.

[0149] For EGFP, the peak of its absorption spectrum is 488nm, which has a minimum difference of 0 with the peak of the excitation light in the 488nm band of the light source, and is less than the threshold of 30. The system determines that EGFP can be excited in the system, and the peak of the excitation light band is 488nm.

[0150] For EYFP, the peak of its absorption spectrum is 513nm, which has a minimum difference of 2 with the peak of the excitation light in the 515nm band of the light source, and is less than the threshold of 30. The system determines that EYFP can be excited in the system, and the peak of the excitation light band is 560nm.

[0151] For ECFP, the peak of its absorption spectrum is 434nm, which has a minimum difference of 1 with the peak of the excitation light in the 435nm band of the light source, and is less than the threshold of 30. The system determines that ECFP can be excited in the system, and the peak of the excitation light band is 560nm.

[0152] For fluorescent probes that can be excited, calculate the brightness of the fluorescent probe in each detection channel.

[0153] From (3.1), we can see that all four fluorescent probes can be excited. Let’s take mCherry as an example for analysis.

[0154] For mCherry, the optical components of the excitation light path are the light source (using excitation light with a peak value of 560nm), the excitation filter, the first dichroic mirror D1 and the objective lens. The intersection of the spectral band range, the reflection band of the dichroic mirror and the passband of the excitation filter is obtained, and the passband of the light path is 548nm-571nm. After obtaining the passband of the light path, the extinction coefficient of the imaging system is calculated to be 928077.

[0155] There are two detection light paths.

[0156] The optical elements of the first detection optical path are objective lens, dichroic mirror D1, emission filter EM1, dichroic mirror D2, emission filter EM2, SCMOS1. The passband of the first detection optical path is 445nm-448nm, 604nm-617nm. After obtaining the passband of the optical path, the photon utilization rate of the first detection optical path of the system is calculated to be 0.047.

[0157] The optical elements of the second detection optical path are objective lens, dichroic mirror D1, emission filter EM1, dichroic mirror D2, emission filter EM3, SCMOS2. The passband of the second detection optical path is 503nm-522nm, 674nm-749nm. After obtaining the passband of the optical path, the photon utilization rate of the second detection optical path of the system is calculated to be 0.022.

[0158] After obtaining the photon utilization rate of different detection channels and the extinction coefficient of the system, in the first detection channel, the brightness value detected by SCMOS1 is 43617, and in the second detection channel, the brightness value detected by SCMOS2 is 20418. The brightness threshold is set to 20000, and the brightness values ​​of both channels are greater than the threshold, that is, mCherry can be imaged in both channels, and SCMOS1 can achieve better imaging effects.

[0159] After the brightness calculation is completed, the system will automatically configure the one-click automatic imaging process of mCherry. When the user selects mCherry as the fluorescent probe, after issuing the imaging command, the system will automatically set the light source to emit excitation light with a peak value of 560nm, start SCMOS1, and collect the fluorescence signal emitted by mCherry on SCMOS1 according to the exposure time and light intensity configured by the user, and save the value locally for the user to view.

[0160] For the four fluorescent probes, the systematically analyzed fluorescent probe imaging quality assessment information is shown in Table 1.

[0161] Table 1

[0162] Fluorescent probes Excitation light peak Channel 1 Brightness Channel 2 Brightness Detector selection Can imaging be performed EGFP 488nm 3394 159421 SCMOS2 able mcherry 560nm 43617 20418 SCMOS1 able ECFP 435nm 0 0 - cannot EYFP 515nm 0 0 - cannot

[0163] 4) For the fluorescent probes capable of imaging, two of them are combined into donor-acceptor pairs and then preliminary FRET imaging screening is performed.

[0164] (1) From 3), it can be determined that the fluorescent probes that can be imaged in the system are EGFP and mCherry.

[0165] For EGFP, the total area of ​​its emission spectrum is calculated to be 65.04, and the total area of ​​its absorption spectrum is 43.67. From 3), it can be determined that the wavelength passbands of its optimal detection channel are 503nm-522nm and 674-749nm, and the detectable band accounts for 17.59 of the total area of ​​the emission spectrum.

[0166] For mCherry, the total area of ​​its emission spectrum is calculated to be 64.18, and the total area of ​​its absorption spectrum is 65.35. From 3), it can be determined that the wavelength passband of its optimal detection channel is 445nm-448nm and 604nm-617nm, and the detectable band accounts for 13.61 of the total area of ​​the emission spectrum.

[0167] Match EGFP and mCherry to check whether they meet the following two conditions.

[0168] ① Set the threshold to 0.2. For EGFP, the ratio of the total area of ​​the band that can be detected under the optimal detection channel to the total area of ​​the emission spectrum is 0.27, which is greater than 0.2, meeting the condition;

[0169] For mCherry, the ratio of the total area of ​​the emission spectrum to the total area of ​​the emission spectrum detected under the optimal detection channel is 0.21, which is greater than 0.2, satisfying the condition.

[0170] ② Set the threshold to 0.3, let EGFP be the donor and mCherry be the acceptor, and calculate that the overlapping area of ​​the mCherry absorption spectrum and the EGFP emission spectrum is 22.30, the total area of ​​the EGFP emission spectrum is 65.04, the total area of ​​the mCherry absorption spectrum is 65.35, and the ratio of the overlapping area of ​​the mCherry absorption spectrum and the EGFP emission spectrum to the total area of ​​the EGFP emission spectrum is 0.34, which is greater than 0.3, which meets the conditions; the ratio of the overlapping area of ​​the mCherry absorption spectrum and the EGFP emission spectrum to the total area of ​​the mCherry absorption spectrum is 0.34, which is greater than 0.3, which meets the conditions.

[0171] After judgment, EGFP and mCherry are determined to be a possible FRET pair, with EGFP as the donor and mCherry as the acceptor. The system saves them in the selection list and saves the relevant camera settings of the two fluorescent probes (including separate imaging settings and E-FRET imaging settings) for users to select and perform fluorescence imaging when performing E-FRET parameter calibration.

[0172] 5) For the donor-acceptor pairs preliminarily screened, a standard E-FRET imaging setting is generated, and then the imaging system is controlled according to the standard E-FRET imaging setting to perform imaging experiments on the standard samples provided by the user, and FRET effect judgment and E-FRET parameter correction are performed accordingly. For the donor-acceptor pairs that are determined to be able to produce a FRET effect, the corresponding E-FRET shooting process is automatically generated; the standard samples include single-transfection donor samples, single-transfection acceptor samples, and samples containing both donors and acceptors.

[0173] For the possible FRET pairs screened in 4), the user is required to provide a single-transfection sample of the corresponding fluorescent probe and a sample containing both the donor and the acceptor for E-FRET parameter calibration.

[0174] For the EGFP-mCherry pair, EGFP is used as the donor and mCherry as the acceptor. The user provides single-transfected EGFP samples, single-transfected mCherry samples, and samples containing both EGFP and mCherry.

[0175] For the single-transfected EGFP sample, the actual image (after contrast enhancement) is as follows Figure 3 As shown, Figure 3The three images in the middle are, from left to right, images collected in channel two with donor excitation light (488nm); images collected in channel one with donor excitation light (488nm); and images collected in channel one with acceptor excitation light (561nm).

[0176] For the single transfection mCherry sample, the actual image (after contrast enhancement) is as follows Figure 4 As shown, Figure 4 The three images in the middle are, from left to right, images collected in channel two with donor excitation light (488nm); images collected in channel one with donor excitation light (488nm); and images collected in channel one with acceptor excitation light (561nm).

[0177] For samples containing both GFP and mCherry, three images were taken according to the standard E-FRET protocol. Figure 5 As shown, Figure 5 The three images in the middle are, from left to right, images collected in channel two with donor excitation light (488nm); images collected in channel one with donor excitation light (488nm); images collected in channel one with acceptor excitation light (561nm).

[0178] Using the above nine images, we can calculate according to the formula:

[0179] d=0.433955, c=0.08367, b=0.002054, a=0.415537; G=1.640552, k=2.984713.

[0180] 6) When the user selects a saved donor-acceptor pair and provides an experimental sample for an experiment, the imaging system is controlled to execute the E-FRET shooting process corresponding to the donor-acceptor pair to perform an imaging experiment on the experimental sample, and the E-FRET result is calculated using the result of the E-FRET parameter calibration.

[0181] After the calculation is completed, the configuration of the above FRET pair, E-FRET correction parameters and E-FRET imaging settings are saved. After saving, the user can directly place the sample corresponding to the FRET pair for a one-click fast E-FRET experiment.

[0182] On the one hand, the control system of the present invention can automatically match the imaging channel according to the fluorescent probe information input by the user. In this regard, the present invention not only proposes a method for matching the imaging channel according to the fluorescent probe, but also provides a preferred channel brightness calculation method. It should be noted that those skilled in the art can also use the method of matching the imaging channel of the fluorescent probe proposed in the present invention to obtain the matching information of the fluorescent probe and the imaging channel, and then directly use this matching information in the multi-channel fluorescence microscopy imaging system to realize the control of automatic imaging according to the fluorescent probe.

[0183] The imaging system, which is the loading object of the control system of the present invention, needs to be equipped with a fully electrically controllable multi-band excitation light source and detector, etc., so as to ensure that the fluorescent probe can be imaged with one click. For the user, it is only necessary to further provide relevant imaging parameters such as excitation light intensity, exposure time and photo-taking instructions, so that the imaging system can allocate the imaging channel with the highest imaging quality to output the imaging results. There is no need for the user to judge the detector and excitation light band required for the imaging channel, etc., which lowers the threshold of fluorescence imaging technology.

[0184] On the other hand, the control system of the present invention provides a fast and stable E-FRET solution, which can automatically match FRET pairs according to the fluorescent probes in the system and provide users with a complete E-FRET method (the entire process from parameter correction to E-FRET shooting). Users only need to provide standard samples of corresponding FRET pairs to complete a one-click E-FRET experiment in this system.

Claims

1. A multi-channel fluorescence microscopy imaging control system based on automatic E-FRET imaging of fluorescent probes, which is applied to a multi-channel fluorescence microscopy imaging system, characterized in that: include: System parameter input software module: determining the relevant information required for establishing connection with each electric control in the imaging system and inputting the optical characteristic parameters of each optical element in the imaging system; Fluorescent probe information input software module: input the optical characteristic parameters of the fluorescent probe; Imaging matching software module: performing automatic imaging matching according to the optical characteristics of the fluorescent probe and the imaging system, and saving the imaging settings of the fluorescent probe; Preliminary FRET imaging screening software module: for the fluorescent probes capable of imaging, two of them are combined into donor-acceptor pairs and then preliminary FRET imaging screening is performed; FRET effect judgment and E-FRET parameter correction software module: for the donor-acceptor pairs preliminarily screened, a standard E-FRET imaging setting is generated, and then the imaging system is controlled according to the standard E-FRET imaging setting to perform imaging experiments on the standard samples provided by the user, and the FRET effect judgment and E-FRET parameter correction are performed accordingly, and for the donor-acceptor pairs determined to be capable of FRET effect, the corresponding E-FRET shooting process is automatically generated and saved; the standard samples include single-transfer donor samples, single-transfer acceptor samples, and samples containing both donors and acceptors; E-FRET imaging execution software module: when the user selects a saved donor-acceptor pair and provides an experimental sample for an experiment, the imaging system is controlled to execute the E-FRET shooting process corresponding to the donor-acceptor pair to perform an imaging experiment on the experimental sample, and the E-FRET result is calculated using the result of the E-FRET parameter correction.

2. The control system according to claim 1, characterized in that: Perform a preliminary FRET imaging screen as follows: 1) According to the normalized spectral characteristics of the fluorescent probe absorption spectrum , calculate the total area of ​​its absorption spectrum ; According to the normalized spectral characteristics of its emission spectrum , and the wavelength passband of the detection channel described in the imaging setup , calculate the total area of ​​its emission spectrum and the detectable wavelengths occupy the total area of ​​the emission spectrum ; 2) If the donor-acceptor pair meets the following two conditions, the pair of fluorescent probes is considered to be a possible FRET pair: ① For both the donor and the acceptor, the ratio of the total area of ​​the detectable band to the total area of ​​the complete emission spectrum must be greater than or equal to the threshold ,Right now ， Threshold It is set according to experience to ensure that the emission light of the fluorescent probe can be detected by the imaging system, that is, to define whether the fluorescent probe can be illuminated in the imaging system; ② For the acceptor, the ratio of the area of ​​the overlapping part of its absorption spectrum and the donor emission spectrum to the total area of ​​the donor emission spectrum, as well as the ratio of the area of ​​the overlapping part of its absorption spectrum and the donor emission spectrum to the total area of ​​the acceptor absorption spectrum, must be greater than or equal to the threshold ,Right now ， ， Threshold Take 0.

3.

3. The control system according to claim 1 or 2, characterized in that: E-FRET parameter calibration mainly refers to calculating the E-FRET correction coefficients a, b, c, d, G, k, which includes the following steps: Calculate the corrected spectral crosstalk coefficients a, b, c, and d of the single-turn donor and single-turn acceptor, where a represents the degree of crosstalk caused by the direct excitation of the acceptor by the donor excitation light, b represents the degree of crosstalk of the emission light of the acceptor directly excites the acceptor by the acceptor excitation light, c represents the degree of crosstalk caused by the direct excitation of the donor by the acceptor excitation light, and d represents the degree of crosstalk of the emission light of the donor directly excites the donor by the donor excitation light. Their calculation formulas are: ， ， The theoretical sensitized emission value Fc is calculated using a sample containing both donor and acceptor: ， It represents the gray value of the detected image, and its subscripts have the following meanings: the first letter represents the excitation light, where D represents the donor excitation light and A represents the acceptor excitation light; the second letter represents the detection channel, where D represents the donor detection channel and A represents the acceptor detection channel; the letters in brackets represent the sample type, where D represents a single-transfer donor and A represents a single-transfer acceptor; DA represents that the sample contains both donors and acceptors; Then the sensitization-quenching conversion factor G and the concentration correction factor k are calculated. The sensitization-quenching conversion factor G is defined as the ratio of the sensitized emission Fc in the DD channel after acceptor photobleaching to the corresponding donor recovery amount. The concentration correction factor k represents the ratio of the donor / acceptor fluorescence intensities of equimolar concentrations in the absence of FRET. The calculation formula is: ， ， Where E is the theoretical FRET efficiency of a sample containing both donor and acceptor.

4. The control system according to claim 1, characterized in that: When automatic imaging matching is performed according to the optical characteristics of the fluorescent probe and the imaging system, an imaging channel is selected according to the brightness of the fluorescent probe in the detection channel of the imaging system, and the imaging channel is selected with a brightness greater than a set threshold detection channel.

5. The control system according to claim 4, characterized in that: Select the detection channel with the highest brightness as the imaging channel.

6. The control system according to claim 4 or 5, characterized in that: The brightness of the fluorescent probe in the detection channel of the imaging system is determined by: Calculate the wavelength passband of the excitation light path of the detection channel ; Calculate the wavelength passband of the detection optical path of the detection channel ; Calculate the extinction coefficient of the excitation light path corrected according to the excitation light source band of the detection channel and the photon utilization rate of the detection optical path , which are defined as: ， ， in, is the molar extinction coefficient of the fluorescent probe; is the normalized spectral characteristic of the emission spectrum of the excitation light source; represents the normalized spectral characteristics of the fluorescent probe absorption spectrum; Indicates the transmittance of the objective lens of the detection channel to light; represents the quantum yield of the fluorescent probe; represents the normalized spectral characteristics of the emission spectrum of the fluorescent probe; The quantum efficiency of the detector of the detection channel to light; Then calculate the brightness of the fluorescent probe in the detection channel , defined as: 。 7. The control system according to claim 6, characterized in that: Before calculating the brightness of the detection channel, the excitation light source band of the detection channel is selected in the following manner: Calculate the absorption spectrum peak of the fluorescent probe The peak of the emission spectrum of the excitation light source of the imaging system The difference and set the threshold Compare and choose The corresponding light source emits light band.

8. The control system according to claim 7, characterized in that: Select the smallest The corresponding light source emits light band.

Images